We propose to develop a rapid, low cost, threshold assay for the detection of HIV infection of HIV vaccination trial participants. Infection by HIV, and viral load are the usual end points for Phase III HIV vaccination trials. Ongoing Phase III HIV vaccine trials require a simple, and low cost molecular diagnostic test that can detect HIV infection in immunized participants. Indeed, current molecular tests are too complex to be used for screening purposes, immunoassays cannot distinguish between immunization and infection, and CD4 cell counts are unlikely to detect early HIV infection. Although FDA requires the use an approved assay to measure viral load in order to meet the demands for licensure, a low cost, moderate complexity, screening test will greatly facilitate these trials. Indeed all participants in both the placebo and vaccine arms must be screened periodically (e.g., at 3 to 6 month intervals). The assay we propose to develop will use our proprietary helicase dependent amplification (HDA) platform as well as a low cost device specifically designed to perform molecular tests without contaminating the laboratory with amplification products. HDA is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. It is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). We propose to use simulated clinical samples generated with the Armored RNA HIV Quant clone spiked into whole blood from HIV seronegative volunteers at known concentrations. Armored RNA will be isolated from plasma prior to RT-HDA testing. Lateral flow devices have already proven their utility in separating nucleic acid amplification products, and are widely used in several moderate complexity and CLIA-waived tests listed in the FDA device database. Detection of amplicons will be accomplished by using latex particles conjugated to antibodies that bind to the probes used to detect the HDA reaction products. The assay uses a sandwich format to detect haptens (biotin, FITC and Digoxigenin) incorporated into one of the HDA primers and into each of the detection probes. The lateral flow strip used for these assays has two capture zones, and thus allows for the detection of one analyte (HIV) as well as a competitive internal control. Assays that fail to give a band in the lateral flow device window are scored as invalid, while assays with a band in the control line alone are scored as true negative. Assays with bands at both the control and test line position are scored as positives at the threshold value (typically 50 copies of the analyte). Assays with a strong band in the test line but no control line are scored as strong positives (typically over 5000 copies of analyte). We will test the specificity and sensitivity of the HIV test with clinical specimens at Vanderbilt University. Kits will be supplied to Dr. Yi-Wei Tang (Vanderbilt University) so his laboratory can evaluate the assays. We may also supply kits to US Government labs that want to try the test. We will also contract with Assuragen to engineer a clone of Armored RNA for our competitive internal control. Finally, we will compare the performance of HDA and the Roche Amplicor HIV-1 DNA PCR (version 1.5) using a set of Dried Blood Spot (DBS) specimens from the CDC's Proficiency Testing (PT) Program. By the end of Phase I, we will have preliminary data for submission of a pre-IDE to the FDA to kick-off a clinical study to seek regulatory clearance for sale of the assay system for human diagnostics using the Roche Amplicor HIV-1 DNA PCR (version 1.5) assay as a predicate device.Narrative More than 90 % of the 40 million human immunodeficiency virus (HIV)-infected persons live in Third World countries (see www.unaids.org/bangkok2004/GAR2004_html/GAR2004_03_en.htm). An HIV vaccine is the lowest cost medical solution for containing the acquired immuno deficiency syndrome (AIDS) epidemic in developing countries. Ongoing Phase III HIV vaccine trials use protection from infection (or reduced viral load when infection still occurs) as an end point. However, in order to meet the demands for licensure the FDA requires us to use an approved assay to measure viral load. These trials require a simple, and low cost molecular diagnostic test that can detect HIV infection in immunized participants. Current evidence suggests that only the central memory pool of CD4 T cells is affected very early in infection. Since this represents less than 1% of the circulating CD4 T cells, existing low cost CD4 monitoring tests used in the third world cannot replace viral RNA as a surrogate for detecting infection. Moreover, rapid immunodiagnostic assays cannot distinguish between immunization and infection, and current molecular diagnostic assays are too complex, and expensive for most developing country laboratories. Although transporting samples, using dry blood spots (DBS), to laboratories that can perform complex molecular tests offers a solution to this problem, a simple, and low cost molecular diagnostic test is preferable from the standpoint of the patient, and medical ethics (i.e., the sooner an infection is detected the sooner the patient could receive treatment with antiviral drugs). Once a participant has been identified as infected, a blood sample can be collected and analyzed with a high complexity, FDA approved viral load assay. BioHelix's proprietary helicase dependent amplification (HDA) platform is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. HDA is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). Indeed, HDA can be performed in a simple $100 water bath. To illustrate the potential of this technology for the third world, we currently sell HDA reagents as part of a teaching kit for use in high schools (sold through Carolina Biological Supply). BioHelix is combining this HDA technology with a low cost (~$2-$3.75) disposable device specifically designed to perform molecular tests without contaminating the laboratory with amplicons. The device uses a lateral flow system to detect amplification products. Lateral flow devices are common in many moderately complex, and CLIA-waived tests listed on the Food and Drug Administration (FDA) device database. We believe this combination of characteristics makes our proposed product ideal for the HIV vaccine trial sites. In addition, recent evidence suggests that more than half of the newly acquired infections are caused by acutely infected individuals who are often seronegative. A low cost nucleic acid screening test will help identify those people and reduce the spread of the HIV epidemic as well as benefit participants in HIV vaccination trials who become infected with the virus. [unreadable] [unreadable] [unreadable]